Process for manufacturing L-(-)-carnitine from a waste product having opposite configuration

ABSTRACT

The present invention relates to a proces for manufacturing L-(-)-carnitine from D-(+)-carnitine or a derivative thereof. D-(+)-carnitine is esterified in order to protect the carboxyl group and subsequently converted to an acyl derivative. The acyl derivative is then converted to a lactone of L-(-)-carnitine. Finally, the lactone is reopened to obtain L-(-)-carnitine.

This is a division of application Ser. No. 08/170,764 filed on Dec. 21,1993 now U.S. Pat. No. 5,412,113.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for manufacturingL-(-)-carnitine from a starting compound containing an asymmetricalcarbon atom having a configuration opposite to that of L-(-)-carnitine.The process of the present invention overcomes the drawbacks ofconventional processes which first convert a starting compound into anachiral intermediate, generally crotonobetaine or gamma-butyrobetaine,and then convert the achiral intermediate to L-(-)-carnitine. Theprocess of the present invention uses D-(+)-carnitine or a derivativethereof as a starting compound.

2. Discussion of the Background

Carnitine contains a single center of asymmetry and therefore exists astwo enantiomers, designated D-(+)-carnitine and L-(-)-carnitine. Ofthese, only L-(-)-carnitine is found in living organisms, where itfunctions as a vehicle for transporting fatty acids across mitochondrialmembranes. Whilst L-(-)-carnitine is the physiologically-activeenantiomer, racemic D,L-carnitine has conventionally been used as atherapeutic agent. It is now recognized, however, that D-(+)-carnitineis a competitive inhibitor of carnitine acyltransferases, and that itdiminishes the level of L-(-)-carnitine in myocardium and skeletalmuscle.

It is therefore essential that only L-(-)-carnitine be administered topatients undergoing haemodialysis treatment or treatment for cardiac orlipid metabolism disorders. The same requirement applies to thetherapeutic utilization of acyl derivatives of carnitine for treatingdisorders of the cerebral metabolism, peripheral neuropathies,peripheral vascular diseases and the like. These disorders are typicallytreated with acetyl L-(-)-carnitine and propionyl L-(-)-carnitine, whichare obtained by acylating L-(-)-carnitine.

Various chemical procedures have been proposed for the industrial-scaleproduction of carnitine. Unfortunately, these procedures are notstereospecific and produce racemic mixtures of D-(+)- and L-(-)-isomers.It is thus necessary to apply resolution methods in order to separatethe enantiomeric constituents of the racemate.

Typically, the D,L-racemic mixture is reacted with an optically activeacid (e.g. D-(-)-tartaric acid, D-(+)-camphorsulfonic acid,(+)-dibenzoyl-D-(-)-tartaric acid, N-acetyl-L-(+)-glutamic acid andD-(+)-camphoric acid) to obtain two diastereoisomers which can beseparated from each other. In the classic process disclosed in U.S. Pat.No. 4,254,053, D-(+)-camphoric acid is used as the resolution agent of aracemic mixture of D,L-carnitinamide, obtaining D-(+)-carnitinamide as aby-product, and L-(-)-carnitinamide which, by hydrolysis, givesL-(-)-carnitine.

However, these resolution procedures are complex and costly, and in allcases result in the production of equimolar quantities ofL-(-)-carnitine and D-(+)-carnitine or a precursor thereof asby-product, having configuration opposite to that of L-(-)-carnitine.Several microbiological processes have recently been proposed forproducing L-(-)-carnitine via stereospecific transformation of achiralderivatives obtained from the huge amounts of D-(+)-carnitine (or of aprecursor thereof, such as D-(+)-carnitinamide) which are generated asby-products in the industrial production of L-(-)-carnitine.

These processes are generally predicated upon the stereospecifichydration of crotonobetaine to L-(-)-carnitine, and differ principallyby virtue of the particular microorganism employed to accomplish thebiotransformation of interest. See, for example, the processes disclosedin: EP 0 12 1444 (HAMARI), EP 0 122 794 (AJINOMOTO), EP 0 148 132(SIGMA-TAU), JP 275689/87 (BIORU), JP 61067494 (SEITETSU), JP 61234794(SEITETSU), JP 61234788 (SEITETSU), JP 61271996 (SEITETSU), JP 61271995(SEITETSU), EP 0 410 430 (LONZA), EP 0 195 944 (LONZA), EP 0 158 194(LONZA), and EP 0 457 735 (SIGMA-TAU).

On the other hand, JP 62044189 (SEITETSU) discloses a process forstereoselectively producing L-(-)-carnitine starting fromgamma-butyrobetaine, which is in turn obtained enzymically fromcrotonobetaine.

All of these processes have several drawbacks. First, D-(+)-carnitinemust first be converted to an achiral compound (crotonobetaine,gamma-butyrobetaine) before it can be used as the starting compound inall of the aforesaid microbiological processes.

In addition, the microbiological procedures proposed to date have notproven practicable for manufacturing L-(-)-carnitine on an industrialscale for one or more of the following reasons:

(i) the yield of L-(-)-carnitine is extremely low;

(ii) the microorganisms must be cultivated in a costly nutritive medium;

(iii) the microorganism can only tolerate low concentrations [up to 2-3%(w/v)] of crotonobetaine;

(iv) side reactions occur, such as the reduction of crotonobetaine togamma-butyrobetaine or the oxidation of L-(-)-carnitine to3-dehydrocarnitine. These side reactions reduce the final yield ofL-(-)-carnitine.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide anefficient method for producing L-(-)-carnitine from a derivative ofD-(+)-carnitine.

The process of the present invention overcomes all of the aforesaiddrawbacks of the known processes, allowing high yields ofL-(-)-carnitine to be obtained starting from a by-product havingconfiguration opposite to that of L-(-)-carnitine with no need to firstconvert the starting by-product into an achiral intermediate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The process of the invention is illustrated in the following reactionscheme: ##STR1##

With reference to the reaction scheme, the D-(+)-carnitinamide salt 1,wherein X is any suitable counterion is hydrolyzed to D-(+)-carnitine 2via conventional procedures (see, for example JP 287065/1989,incorporated herein by reference). X is suitably a halogen, preferablychloride; phosphate; perchlorate; metaperiodate; tetraphenylborate; analkylsulfonate having 1-12 carbon atoms, preferably dodecylsulphonate;trifluoroacetate; tetrahalogenborate; fumarate or an alkylsulphatehaving 10-14 carbon atoms.

D-(+)-carnitine 2 is then converted to the ester 3 in order to protectthe carboxyl group. Suitable esters 3 are those wherein R₁ is (1) astraight or branched alkoxy group having 1-11 carbon atoms or (2) anarylalkoxy or diarylalkoxy group wherein the aryl is a monocyclic orbicyclic aryl and the alkyl has 1-4 carbon atoms. Suitable monocyclic orbicyclic aryl groups contain 5-12 carbon atoms and can be optionallysubstituted with a lower alkyl group having 1-4 carbon atoms; an alkoxygroup having 1-4 carbon atoms; halogen, preferably fluorine or chlorine;a nitro group or an amino group. Suitable arylalkoxy or diarylalkoxygroups include p-methoxybenzyloxy, 1-naphthalenemethoxy,2-naphthalenemethoxy, and diphenylmethoxy. A particularly preferredarylalkoxy group is benzyloxy.

The esterification of 2 to 3 is carried out via conventional procedures.For instance, when R₁ is benzyloxy, the preparation of D-(+)-carnitinebenzyl ester is carried out as disclosed in Biochim. Biophys. Acta(1967) 137:98, incorporated herein by reference.

The ester 3 is then converted to the acyl derivative 4. Y, which can bethe same as X, is preferably a counterion imparting solubility to 4. ORis a leaving group wherein R is an alkylsulfonyl group having 1-12carbon atoms, formyl or trifluoroacetyl. Preferably, the alkylsulfonylgroup is selected from methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), p-bromobenzenesulfonyl (brosyl), p-nitrobenzenesulfonyl(nosyl), trifluoromethanesulfonyl (triflyl), nonafluoromethanesulfonyl(nonaflyl) and 2,2,2-trifluoroethanesulfonyl (tresyl). Mesyl isparticularly preferred.

The acylation of 3 to 4 is carried out by reacting the ester 3 with anacylating agent RY wherein Y is halogen, or RY itself is an anhydrideand R is an acyl group as defined above. Preferably RY is the chlorideof the selected acyl group.

The acylation reaction is suitably carried out in pyridine,alkylpyridines, or other basic solvents such as triethylamine or in amixture of an anhydrous, inert organic solvent such as acetonitrile ormethylene chloride with a base such as pyridine, lutidine, picoline orpolyvinylpyridine.

The acylating agent is suitably added at ratios ranging from 1:1 to1:10, preferably 1:3. The resulting reaction mixture is kept understirring at temperatures comprised between 0° C. and 50° C. for 1-24hours Compound 4 is isolated by precipitation with a suitable solventsuch as ethyl ether or hexane and purified by dissolving it in water andextracting with an organic solvent.

The carboxyl group is restored into compound 4 via known procedures, toyield acyl D-(+)-carnitine 5. In some instances, if needed, compound 4is subjected to hydrogenation.

Hydrogenation of 4 is suitably carried out in an aqueous solution, at pH2-4, or in methanol at 0° C.-25° C., for 1-8 hours, at 1-4 hydrogenatmospheres, in the presence of a hydrogenation catalyst such as 5% or10% Pd/C. Acyl D-(+)-carnitine 5 can be isolated by filtering off thecatalyst and lyophilizing or concentrating the aqueous solution.

Acyl D-(+)-carnitine 5 is then converted to the lactone 6 ofL-(-)-carnitine. The lactonization is suitably carried out in an aqueousbasic environment: either with NaHCO₃ (ratio 1:1) or with an AMBERLITEIRA-402 (manufactured by Rohm & Haas Co., GERMANY) basic resin activatedin HCO₃ ⁻ form or with an LA2 resin (Rohm & Haas). The lactone isisolated by evaporating the aqueous solution or precipitating it as asalt (for example, as tetraphenylborate or reineckate).

Finally, lactone 6 is suitably converted to L-(-)-carnitine inner salt7. The lactone is dissolved in water and the resulting solution treatedwith a base such as NaHCO₃ (ratio 1:1), for 8-24 hours.

L-(-)-carnitine can suitably be purified from the salts which are formedfrom the X⁻ anion, from the excess, if any, of the acyl halogenide, frompyridine, and the like, by chromatographing the aqueous solution on astrongly acidic resin such as IR 120 (Rohm & Haas), eluting with waterand then with NH₄ OH, or alternatively eluting first on a strongly basicresin such as AMBERLITE IRA 402 (Rohm & Haas) activated in OH form andthereafter on a weakly acid resin such as AMBERLITE IRC-50 (Rohm &Haas).

It should be understood that, whereas the process disclosed above hasbeen described, for the sake of clarity, as a sequence of six distinctoperating steps, the corresponding industrial process consists of foursteps only. When the process of the present invention is carried out asan industrial process, the acyl D-(+)-carnitine ester 4 can be directlyconverted to L-(-)-carnitine inner salt 7 without isolating either theacyl D-(+)-carnitine 5 or the lactone 6.

In fact, the ester of acyl D-(+)-carnitine 4 is hydrogenated and thehydrogenation catalyst filtered off. The resulting aqueous solution isbrought to pH 7-9, preferably 8-9 and kept at this pH value for 30-50hours yielding L-(-)-carnitine. L-(-)-carnitine thus obtained ispurified by removing the salts by treatment with acidic and basicresins.

In the following example which describes one embodiment of the processof the invention, the intermediate compounds 4, 5 and 6 were isolated soas to exhaustively characterize them from a physico-chemical standpoint,insofar as these intermediates are novel compounds.

It will be, however, apparent to any expert in organic synthesis thatthe industrial process comprises the following steps only:

(a) hydrolysis of D-(+)-carnitinamide 1 to D-(+)-carnitine 2;

(b) esterification of D-(+)-carnitine 2 to the ester 3 to protect thecarboxyl group;

(c) acylation of the hydroxyl group of ester 3 with an acylating agentRY wherein Y is a halogen or RY itself an anhydride, with the resultingformation of a leaving group OR wherein R has the previously definedmeanings, thus obtaining the ester 4 of D-(+)-carnitine; and

(d) conversion of 4 to L-(-)-carnitine inner salt 7.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

In the following examples, the conversion of D-(+)-carnitinamide toD-(+)-carnitine and the conversion of the latter compound to ester 3 arenot described for the sake of brevity and since those conversions can becarried out via procedures well-known to any expert in organicsynthesis.

Moreover, with reference to the numbering of the compound show in thereaction scheme, the lower-case letters "a", "b" and "c" are used in theexample to indicate X⁻ =perchlorate, chloride and methanesulfonate,respectively.

EXAMPLE Preparation of Methanesulfonyl D-(+)-carnitine Benzyl EsterPerchlorate (4a)

Methanesulfonyl chloride (25.77 g; 225 mmoles) was added in the space offive minutes to a solution of D-(+)-carnitine benzylester perchlorate(24.4 g; 75 mmoles) in anhydrous pyridine (100 mL) cooled in an icebath. At the end of the addition, the solution was kept under stirringat room temperature for 1 hour and 45 minutes. The solution was thenpoured into an Erlenmeyer flask containing 500 mL Et₂ O under stirring.

The oily precipitate obtained by decantation of Et₂ O was taken up withCH₂ Cl₂ (300 mL), that solution was washed with 2N HCl (4×5 mL),saturated solution of NaCl (1×20 mL) and dried over anhydrous Na₂ SO₄.

Following evaporation of the organic phase, 22 g of an amorphous solidwere obtained. Yield 70%. Differential thermal analysis: it decomposesat about 180° C. [α]_(D) ²⁵ =+20.0° [c=1% MeOH)

    ______________________________________                                        TLC = silica gel                                                                         Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH                                         42/28/7/10.5/10.5                                                    Rf = 0.5                                                           ______________________________________                                        Elementary analysis for C.sub.15 H.sub.24 ClNO.sub.9 S                                    C %    H %        N %  Cl %                                       ______________________________________                                        Calculated  41.91  5.63       3.25 8.25                                       Found       41.81  4.72       3.28 8.10                                       ______________________________________                                    

¹ H NMR ((CD₃)₂ CO): δ 7.45-7.30 (m, 5H, aromatics); 5.71-5.62 (m, 1H,--CHOMs); 5.20 (s, 2H, --CH₂ Ph); 4.24-4.02 (m, 2H, --CH₂ N⁺ Me₃); 3.47(s, 9H, --N⁺ Me₃); 3.30 (s, 3H, CH₃ SO₃ --) 3.20 (2H, d, --CH₂ COO⁻) ¹³C NMR ((CD₃)₂ CO): δ 169.413; 136.685; 129.153; 71.902 67.496; 54.683;39.387; 38.640 IR (KBr)=υ(cm⁻¹) 1735 (--C═O), 1341 and 1174 (CH₃ SO₃ --)

HPLC

Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm

Eluant=CH₃ CN/KH₂ PO₄ 50 mM (65/35) pH=3.5 with H₃ PO₄

Flow rate=0.75 ml/min

Retention time=9.35 min

Detector=RI Waters 410

Preparation of Methanesulfonyl D-(+)-carnitine Benzyl Ester Chloride(4b)

18.3 g (42.6 mmoles) of methanesulfonyl D-(+)-carnitine benzyl esterperchlorate were dissolved in 300 mL CH₃ OH and few mL CH₃ CN (tillcomplete dissolution). The solution thus obtained was percolated throughAMBERLYST A-21 resin (300 g) activated by percolating therethrough 1NHCl, then H₂ O till neutrality and finally CH₃ OH. Following methanolevaporation, 15.5 of a solid product were obtained. Yield: quantitative.Differential thermal analysis: it decomposes at about 150° C. [α]_(D) ²⁵=+22.6° [c=1% MeOH)

    ______________________________________                                        TLC = silica gel                                                                         Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH                                         42/28/7/10.5/10.5                                                    Rf = 0.5                                                           ______________________________________                                        Elementary analysis for C.sub.15 H.sub.24 ClNO.sub.5 S                                        C %     H %     N %   Cl %                                    ______________________________________                                        Calculated (+3.3% di.H.sub.2 O)                                                               47.62   6.76    3.70  9.37                                    Found           47.88   7.52    3.77  9.04                                    ______________________________________                                    

¹ H NMR (D₂ O): δ 7.50-7.45 (m, 5H, aromatics); 5.70-5.62 (m, 1H,--CHOMs); 5.40-5.30 (m, 2H, --CH₂ Ph); 4.03-3.72 (m, 2H, --CH₂ N⁺ Me₃);3.25 (s, 3H, CH₃ SO₃ --) 3.22 (s, 9H --N⁺ Me₃); 3.15 (2H, d, --CH₂ COO⁻)¹³ C NMR (D₂ O): δ 172.789; 137.950; 131.695; 73.929; 70.651; 56.831;41.475; 40.920 IR (pure)=υ(cm⁻¹) 1734 (--C═O), 1340 and 1174 (CH₃ SO₃ ⁻)

HPLC

Column Nucleosil 5-SA; diameter=4 mm; length=200 mm

Eluant CH₃ CN/KH₂ PO₄ 50 mM (65/35) pH=3.5 with H₃ PO₄

Flow rate 0.75 ml/min

Retention time=9.41 min

Detector=RI Waters 410

Preparation of Methanesulfonyl D-(+)-carnitine Perchlorate (5a)

10% Pd/C (300 mg) was added to a solution of methanesulfonylD-(+)-carnitine benzyl ester perchlorate (3.0 g; 7 mmoles) in CH₃ OH (50mL).

The resulting mixture was kept under stirring in a hydrogen atmosphereat 45 p.s.i. (219.7 kg/m²) in a Parr apparatus for 4 hours. After thecatalyst was filtered off and the solvent evaporated, 2.3 g of a whitesolid product were obtained. Yield: quantitative. Differential thermalanalysis: incipient decomposition at about 170° C. [α]_(D) ²⁵ =+19.6°[c=1% MeOH)

    ______________________________________                                        TLC = silica gel                                                                         Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH                                         42/20/7/10.5/10.5                                                  Rf = 0.15                                                            ______________________________________                                        Elementary analysis for C.sub.8 H.sub.18 ClNO.sub.9 S                                     C %    H %        N %  Cl %                                       ______________________________________                                        Calculated  28.28  5.34       4.12 10.43                                      Found       28.78  5.34       4.15 10.23                                      ______________________________________                                    

¹ H NMR (D₂ O): δ 5.68-5.59 (m, 1H, --CHOMs, ); 4.05-3.75 (m, 2H, --CH₂N⁺ Me₃); 3.33 (s, 3H, CH₃ SO₃ --) 3.27 (s, 9H-N⁺ Me₃); 3.15-3.00 (m, 2H,--CH₂ COOH) ¹³ C NMR (D₂ O): δ 175.192; 74.423; 70.838; 56.971; 41.662;40.774 IR (KBr)=υ(cm⁻¹) 1731 (C═O), 1340 and 1174 (CH₃ SO₃ --)

HPLC

Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm

Eluant=CH₃ CN/KH₂ PO₄ 50 mM (65/35) pH=3.5 with H₃ PO₄

Flow rate=0.75 ml/min

Retention time=11.33 min

Detector=RI Waters 410

Preparation of Methanesulfonyl D-(+)-carnitine Chloride (5b)

10% Pd/C (500 mg) was added to a solution of methanesulfonylD-(+)-carnitine benzyl ester chloride (5.1 g; 13.9 mmoles) in H₂ O (60mL) acidified to pH 4 with 1N HCl. The resulting mixture was kept understirring in a hydrogen atmosphere, at 45 p.s.i. (219.7 kg/m²) in a Parrapparatus for 4 hours.

The catalyst was filtered off and the aqueous solution lyophilized,giving 3.8 g of a white solid product. Yield: quantitative. Differentialthermal analysis: it decomposes at about 150° C. [α]_(D) ²⁵ =+29.5°[c=1% H₂ O)

    ______________________________________                                        TCL = silica gel                                                                         Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH                                         42/20/7/10.5/10.5                                                  Rf = 0.15                                                            ______________________________________                                        Elementary analysis for C.sub.8 H.sub.18 ClNO.sub.5 S                                     C %    H %        N %  Cl %                                       ______________________________________                                        Calculated  34.84  6.58       5.10 12.86                                      Found       35.37  6.82       5.24 12.45                                      ______________________________________                                    

¹ H NMR (D₂ O): δ 5.70-5.60 (m, 1H, --CHOMs, ); 4.06-3.75 (m, 1H, --CH₂N⁺ Me₃); 3.33 (s, 3H, CH₃ SO₃ ⁻) 3.27 (s, 9H --N⁺ Me₃); 3.15-3.00 (m,2H, --CH₂ COOH) ¹³ C NMR (D₂ O): δ 175.326; 74.530; 70.851; 56.964;41.668; 40.914 IR (KBr)=υ(cm⁻¹) 1720 (C═O), 1335 and 1175 (CH₃ SO₃ --)

HPLC

Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm

Eluant=CH₃ CN/KH₂ PO₄ 50 mM (65/35) pH =3.5 with H₃ PO₄

Flow rate=0.75 ml/min

Retention time=11.38 min

Detector=RI Waters 410

Preparation of the Lactone of L-(-)-carnitine Chloride (6b)

NaHCO₃ (0.46 g; 5.4 mmoles) was added to a solution of methanesulfonylD-(+)-carnitine chloride (1.5 g; 5.4 mmoles) in H₂ O (25 mL) and theresulting solution was kept under stirring for 20 hours. The solutionwas then lyophilized, the residue taken up with CH₃ CN and theundissolved solid filtered off. Following solvent evaporation, 0.98 g,of the title compound were obtained. Yield: quantitative.

    ______________________________________                                        TLC = silica gel                                                                         Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH                                         42/28/7/10.5/10.5                                                    Rf = 0.1                                                           ______________________________________                                    

¹ NMR (D₂ O): δ 5.33-5.24 (m, 1H, --CHOCO--); 3.96-3.88 (m, 3H), --CH₂N⁺ Me₃, --CHHCO--); 3.53-3.44 (m, 1H, --CHHCOO--); 3.24 (s, 9H, --N⁺Me₃) ¹³ C NMR (D₂ O): δ 172.428; 70.671; 68.094; 56.991; 41.394 IR(KBr)=υ(cm⁻¹) 1850 (C═O)

HPLC

Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm

Eluant=CH₃ CN/KH₂ PO₄ 50 mM (65/35) pH=3.5 with H3PO₄

Flow rate=0.75 ml/min

Retention time=19.23 min

Detector=RI Waters 410

Preparation of the Lactone of L-(-)-carnitine Methanesulfonate (6c)

An aqueous solution of methanesulfonyl D-(+)-carnitine chloride (1.5 g;5.4 mmoles) was perchlorated through an IRA-402 resin (30 g) activatedto HCO₃ ⁻ form and cooled to 5° C., eluting with water at 5° C. tillcomplete elution (controlled by TCL). The eluate was kept at roomtemperature for 4 hours. Following evaporation of the aqueous solution,1.3 g of a raw product which was taken up with CH₃ CN, were obtained.Evaporation of the organic solvent yielded 1 g of a white solid. Yield:80% Differential thermal analysis: incipient decomposition at 160° C.[α]_(D) ²⁵ =+24.7° (c=1% MeOH)

    ______________________________________                                        TCL = silica gel                                                                         Eluant = CHCl.sub.3 /MeOH/iPrOH/H.sub.2 O/AcOH                                         42/28/7/10.5/10.5                                                    Rf = 0.1                                                           ______________________________________                                        Elementary analysis for C.sub.8 H.sub.17 NO.sub.5 S                                      C %          H %    N %                                            ______________________________________                                        Calculated 40.16        7.16   5.85                                           Found      39.61        7.13   5.77                                           ______________________________________                                    

¹ H NMR (D₂ O): δ 5.35-5.25 (m, 1H, --CHOCO--); 3.98-3.89 (m, 3H, --CH₂N⁺ Me₃, --CHHCOO--), 3.54-3.46 (m, 1H, --CHHCOO--); 3.26 (s, 9H, --N⁺Me₃); 2.81 (s, 3H, CH₃ SO₃ --) ¹³ C NMR (D₂ O): δ 172.428; 70.671;68.094; 56.991; 45.320; 41.394 IR (KBr)=υ(cm⁻¹) 1835 (C═O)

HPLC

Column=Nucleosil 5-SA; diameter=4 mm; length=200 mm

Eluant=CH₃ CN/KH₃ PO₄ 50 mM (65/35) pH=3.5 with H₃ PO₄

Flow rate=0.75 ml/min

Retention time=19.48 min

Detector=RI Waters 410

Preparation of L-carnitine Inner Salt (7) from the Lactone ofL-(-)-carnitine Methanesulfonate (6c)

NaHCO₃ (0.34 g; 4 mmoles) was added to a solution of the lactone ofL-(-)-carnitine methanesulfonate (0.96 g; 4 mmoles) in H₂ O (20 mL) andthe resulting solution was kept under stirring at room temperature for20 hours. The solution was then percolated through AMBERLITE IR-120resin (20 g) eluting first with water till neutrality to removemethanesulfonic acid and then with 2% NH₃ aqueous solution collectingthe eluate till complete elution of L-(-)-carnitine inner salt(controlled by TLC).

Following evaporation of the aqueous solution, 0.64 g of L-(-)-carnitineinner salt were obtained.

Alternatively, the reaction mixture was percolated through IRA-402 resin(20 g) activated to OH⁻ form, eluting with H₂ O till neutrality. Theeluate was then percolated through IRC-50 resin (20 g) till completeelution of L-carnitine inner salt (controlled by TLC). Followingevaporation of the aqueous solution, 0.64 g of L-(-)-carnitine innersalt were obtained.

Yield: quantitative

The enantiomeric excess (e.e.) was assessed via the following HPLCmethod, after L-(-)-carnitine was derivatized with a chiral reagent. Aschiral reagent, (+)-1-(9-fluorenyl) ethyl chloroformate (FLEC) was used.

column: Nova-pak C₁₈ (4μ) Cartridge

length: 100 mm

diameter: 5.0 mm

Eluant:

Solution A: 5mM tetrabutylammonium hydroxide (TBA⁺ OH⁻), 50 mM KH₂ PO₄75 mL Acetonitrile 25 mL brought to pH 7 with 1N KOH

Solution B: Acetonitrile 75 mL 5 mM KH₂ PO₄ 25 mL

    ______________________________________                                        Elution schedule                                                              Time            % A    % B                                                    ______________________________________                                        0               100    0                                                      15              100    0                                                      16               0     100                                                    22               0     100                                                    23              100    0                                                      30              stop                                                          ______________________________________                                        Detector = Perkin-Elmer Fluorimeter                                                                 Excitation = 260 nm                                                           Slit = 10 nm                                                                  Emission = 315 nm                                                             Slit = 5 nm                                             ______________________________________                                    

L-(-)-carnitine had previously been derivatized with FLEC via thefollowing method:

50 μL of L-(-)-carnitine solution (prepared by dissolving 10 mgcarnitine in 50 mL of 50 mM TBA^(+OH) ⁻ brought to pH 7 withconcentrated H₃ PO₄) and 200 μL of solution consisting of 1 mL FLEC in 3mL acetone, were kept under stirring at 80° C. for 20 minutes.

The solution was cooled and 4 mL of solution A were added thereto, 5 μLof the resulting solution were injected L-(-)-carnitine K¹ =5.79D-(+)-carnitine K¹ =4.82, absent ##EQU1##

Preparation of L-carnitine Inner Salt (7) fromMethanesulfonyl-D-carnitine Chloride (5b)

NaHCO₃ (0.46 g; 5.4 mmoles) was added to a solution ofmethanesulfonyl-D-carnitine chloride (1.5 g; 5.4 mmoles) in H₂ O (25 mL)and the resulting solution was kept under stirring at room temperaturefor 20 hours. Further NaHCO₃ (0.46; 5.4 mmoles) was then added and thesolution was kept under stirring at room temperature for further 20hours. The title compound was isolated as previously described for theisolation of 7 from 6 b.

L-carnitine is obtained from methanesulfonyl-D-carnitine through theformation of the lactone 6, as evidenced by NMR, HPLC, IR and TLCanalysis carried out on a sample obtained by lyophilizing a portion ofthe solution 20 hours following first NaHCO₃ addition.

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and is desired to be secured by Letters Patent ofthe United States is:
 1. A process for producing L-(-)-carnitine innersalt from D-(+)-carnitinamide which comprises:(a) hydrolyzing a salt ofD-(+)-carnitinamide 1 of the general formula ##STR2## wherein X⁻ is anymonovalent counterion to obtain D-(+)-carnitine 2 ##STR3## (b)esterifying said D-(+)-carnitine 2 to an ester 3 of the general formula##STR4## wherein R₁ is (i) a straight or branched alkoxy group having1-11 carbon atoms or (ii) an arylalkoxy or diarylalkoxy group whereinthe aryl group is a monocyclic or bicyclic aryl group containing 5 to 12carbon atoms and the alkyl group has 1-4 carbon atoms, and wherein saidarylalkoxy or diarylalkoxy groups can be optionally substituted with alower alkyl group having 1-4 carbon atoms, an alkoxy group having 1-4carbon atoms, halogen, a nitro group or an amino group; (c) acylatingsaid ester 3 to an acyl derivative 4 of the general formula ##STR5##wherein Y⁻, which is the same as or different than X⁻, is a counterionimparting solubility to 4 and OR is a leaving group wherein R isselected from an alkylsulfonyl having 1-12 carbon atoms, formyl andtrifluoroacetyl, by reacting 3 with an acylating agent (1) of theformula RY wherein Y is a halogen or (2) an anhydride precursor for R,and R has the above defined meaning, with an organic base, in a basicsolvent or in at least one inert organic solvent, at 0° C.-50° C., for1-24 hours; (d) converting the COR₁ group of said acylderivative 4 to acarboxylic group, to obtain an acyl D-(+)-carnitine 5 of the formula##STR6## (e) lactonizing said acyl D-(+)-carnitine 5 to a lactone 6 ofL-(-)-carnitine of the formula ##STR7## by treating 5 in a basicenvironment, and (f) converting said lactone 6 to L-(-)-carnitine innersalt by treating 6 in a basic solution and isolating saidL-(-)-carnitine inner salt.
 2. The process of claim 1, wherein step (d)comprises hydrogenating said acylderivative 4 in an aqueous solution atpH 2-4, at 0° C.-25° C., for 1-8 hours, at 1-4 hydrogen atmospheres, inthe presence of a hydrogenation catalyst.
 3. The process of claim 1,wherein said steps (d), (e) and (f) are carried out as a single step,without isolating said intermediate compounds 5 and
 6. 4. The process ofclaim 1, wherein:X is a halogen, phosphate, perchlorate, metaperiodate,tetraphenylborate, or alkylsulfonate having 1-12 carbon atoms; R₁ isbenzyloxy; and R is methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),p-bromobenzenesulfonyl (brosyl), p-nitrobenzenesulfonyl (nosyl),trifluoromethanesulfonyl (triflyl), nonafluoromethanesulfonyl.(nonaflyl)or 2,2,2-trifluoroethansulfonyl (tresyl).
 5. A process for producingL-(-)-carnitine comprising(a) esterifying D-(+)-carnitine to an ester 3of the general formula ##STR8## wherein R₁ is (i) a straight or branchedalkoxy group having 1-11 carbon atoms or (ii) an arylalkoxy ordiarylalkoxy group wherein the aryl group is a monocyclic or bicyclicaryl group containing 5 to 12 carbon atoms and the alkyl group has 1-4carbon atoms, and wherein said arylalkoxy or diarylalkoxy groups can beoptionally substituted with a lower alkyl group having 1-4 carbon atoms,an alkoxy group having 1-4 carbon atoms, halogen, a nitro group or anamino group; (b) acylating said ester 3 to an acyl derivative 4 of thegeneral formula ##STR9## wherein Y⁻, which is the same as or differentthan X⁻, is a counterion imparting solubility to 4 and OR is a leavinggroup wherein R is selected from an alkylsulfonyl having 1-12 carbonatoms, formyl and trifluoroacetyl, by reacting 3 with an acylating agentof the formula RY wherein Y is a halogen or RY is an anhydride and R hasthe above defined meaning, with an organic base, in a basic solvent orin at least one inert organic solvent, at 0° C.-50° C., for 1-24 hours;(c) converting the COR₁ group of said acylderivative 4 to a carboxylicgroup, to obtain an acyl D-(+)-carnitine 5 of the formula ##STR10## (d)lactonizing said acyl D-(+)-carnitine S to a lactone 6 ofL-(-)-carnitine of the formula ##STR11## by treating 5 in a basicenvironment, and (e) converting said lactone 6 to L-(-)-carnitine bytreating 6 in a basic solution and isolating L-(-)-carnitine inner salt.6. The process of claim 5, wherein steps (c), (d), and (e) are carriedout as a single step, without isolating said intermediate compounds 5and 6.